Keratins are the most abundant proteins in surface epithelia, in which they occur as a cytoplasmic network of 10 nm wide intermediate filaments (IFs). Keratins are encoded by an evolutionarily conserved multigene family, with 54 individual members subdivided into two major types (I and II). The pairwise regulation of type I and type II keratin genes in epithelia reflects a strict heteropolymerization requirement. In addition, keratin genes are regulated in an epithelial tissue-type and differentiation-specific fashion, the functional basis of which is only partly understood. A major role fulfilled by keratin IFs in vivo s to act as a resilient scaffold that endows epithelial cells with the ability to sustain mechanical and non-mechanical stresses. Inherited mutations affecting the coding sequence of keratins account for a large number of epithelial fragility disorders. Several additional functions, which are non-mechanical in nature and manifested in a keratin- and context-dependent fashion, have been identified by us and other researchers in recent years. Here we seek to further our understanding of the mechanisms underlying the structural support function of keratins by focusing on Krt5-Krt14 filaments, which occur in progenitor basal keratinocytes of epidermis and are causally mutated in the disease Epidermolysis Bullosa Simplex (EBS). During the last period of support we investigated the remarkable property of self-organization of Krt5-Krt14 filaments, in vitro and in vivo, and succeeded in crystallizing, for the first time, a portion of te interacting Krt5-Krt14 rod domains. The resulting atomic structure revealed a role for disulfide binding in organizing Krt5-Krt14 IFs into a perinuclear cage in epidermal keratinocytes, with an impact on the size and shape of the nucleus. Going forward we posit that the Krt5-Krt14 pairing is amenable to further crystallization and structure determination (we already determined a second Krt5-Krt14 structure), with unprecedented insight for keratin IF properties and its defect in EBS and related genodermatoses. Moreover, we also posit that intracellular, inter-keratin disulfide bonding represents a major mechanism to regulate the organization of keratin IFs in vivo, determine the filament lifespan (i.e., half- life), and regulate nuclear architecture and keratinocyte differentiation in vivo. In Aim 1, we will: (a) relate specific disulfide bonds involvng Krt5 and Krt14 to specific spatial configurations of IFs in keratinocytes; (b) identify the determinants of keratin filament half-life in epidermal keratinocytes, with an emphasis on disulfide bonding; and (c) assess the functional importance of inter-keratin disulfide bonding in mouse skin tissue. In Aim 2, we will use X-ray crystallography to determine the atomic structure of (a) the heterodimer and (b) the heterotetramer of Krt5 and Krt14 rod domains. Success with this latter effort will help define how EBS-causing mutations impact the structure and properties of Krt5-Kr14 subunits. The proposed research is highly original, addresses crippling knowledge deficiencies in the field, and is poised to deepen our understanding of the mechanistic underpinnings of the clinically-relevant structural support function of keratin filaments in vivo.